Gene-expression profiling of endomyocardial biopsies from dogs with dilated cardiomyopathy phenotype.

INTRODUCTION: The employment of advanced molecular biology technologies has expanded the diagnostic investigation of cardiomyopathies in dogs; these technologies have predominantly been performed on postmortem samples, although the recent use of endomyocardial biopsy in living dogs has enabled a better premortem diagnostic approach to study the myocardial injury. ANIMALS, MATERIALS, AND METHODS: Endomyocardial biopsies were collected in nine dogs with a dilated cardiomyopathy phenotype (DCM-p) and congestive heart failure and submitted to histologic examination, next-generation sequencing (NGS), and polymerase chain reaction analysis. Data from three healthy dogs (Fastq files) were retrieved from a previously approved study and used as a control group for ribonucleic acid sequencing. RESULTS: Histologic examination revealed endocardial fibrosis in six of nine dogs, whereas lymphocytic interstitial infiltrates were detected in two of nine dogs, and lymphoplasmacytic and macrophage infiltrates were detected in one of nine dogs. On polymerase chain reaction analysis, two dogs tested positive for canine parvovirus two and one dog for canine distemper virus. Gene-expression pathways involved in cellular energy metabolism (especially carbohydrates-insulin) and cardiac structural proteins were different in all DCM-p dogs compared to those in the control group. When dogs with lymphocytic interstitial infiltrates were compared to those in the control group, NGS analysis revealed the predominant role of genes related to inflammation and pathogen infection. CONCLUSIONS: Next-generation sequencing technology performed on in vivo endomyocardial biopsies has identified different molecular and genetic factors that could play a role in the development and/or progression of DCM-p in dogs.

Viral load, tissue distribution and histopathological lesions in goats naturally and experimentally infected with the Small Ruminant Lentivirus Genotype E (subtype E1 Roccaverano strain).

Small Ruminant Lentivirus (SRLV) subtype E1, also known as Roccaverano strain, is considered a low pathogenic virus on the basis of natural genetic deletions, in vitro properties and on-farm observations. In order to gain more knowledge on this atypical lentivirus we investigated the in vivo tropism of Roccaverano strain in both, experimentally and naturally infected goats. Antibody responses were monitored as well as tissue distribution and viral load, evaluated by real time PCR on single spliced (gag/env) and multiple spliced (rev) RNA targets respectively, that were compared to histopathological lesions. Lymph nodes, spleen, alveolar macrophages and mammary gland turned out to be the main tissue reservoirs of genotype E1-provirus. Moreover, mammary gland and/or mammary lymph nodes acted as active replication sites in dairy goats, supporting the lactogenic transmission of this virus. Notably, a direct association between viral load and concomitant infection or inflammatory processes was evident within organs such as spleen, lung and testis. Our results validate the low pathogenicity designation of SRLV genotype E1 in vivo, and confirm the monocyte-macrophage cell lineage as the main virus reservoir of this genotype. Accordingly, SRLV genotype E displays a tropism towards all tissues characterized by an abundant presence of these cells, either for their own anatomical structure or for an occasional infectious/inflammatory status.

Antibacterial activities of Manuka and Honeydew honey-based membranes against bacteria that cause wound infections in animals.

INTRODUCTION: In this study, membranes composed of honey (Manuka or Honeydew) and pectin were developed, and the ISO 22196 method was used to evaluate their antibacterial activities against multidrug-resistant bacteria (i.e., Staphylococcus pseudointermedius, Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa) that cause wound infection in animals. The results demonstrated that both Manuka and Honeydew honey-based membranes had strong antibacterial activities against the strain of methicillin-resistant S. pseudointermedius tested. Specifically, membranes composed of Manuka honey were effective in inhibiting the growth of Gram-negative bacteria within 3 h, whereas those composed of Honeydew honey needed 24 h to neutralise bacterial growth. The antimicrobial activities of both membranes developed in this study suggest that they can be effectively used as wound dressing in veterinary clinical medicine.

Dans le cadre de cette étude, on a fabriqué des membranes à base de miel (miel de Manuka et miel de miellat) et de pectine et on a testé, selon le processus ISO 22196, leur activité antibactérienne sur des germes multirésistants provenant de blessures d'animaux (Staphylococcus pseudointermedius, E. coli, Proteus mirabilis und Pseudomonas aeruginosa). Les résultats montrent que les deux types de membranes ont une forte activité bactéricide sur les souches de Staphylococcus pseudointermedius résistantes à la méthicilline. Les membranes à base de miel de Manuka étaient également actives contre tous les germes gram négatifs ét réduisaient leur nombre en 3 heures, alors qu'un contact de 24 heures était nécessaire pour que les membranes à base de miel de miellat réduisent la croissance bactérienne. L'activité antibactérienne des membranes utilisées dans la présente étude justifie leur emploi dans la médecine vétérinaire clinique.

Bartonella-associated inflammatory cardiomyopathy in a dog.

A 6-year-old, male, mongrel dog was presented for acute onset of dyspnea and cough. At admission, the dog was cachectic and severely depressed. The electrocardiogram showed a sinus rhythm conducted with left bundle truncular branch block and interrupted by frequent multiform ventricular ectopic beats organized in allorhythmias. Thoracic radiographs revealed a marked cardiomegaly with perihilar edema, whereas transthoracic echocardiography revealed a dilated cardiomyopathy with segmental dyskinesis. Furosemide, enalapril, pimobendan, and mexiletine were prescribed, and a Holter was scheduled after resolution of congestive heart failure. Three days later, the dog died suddenly during sleep. Histopathology revealed diffuse myocyte hypertrophy with multifocal hemorrhages, alternating to areas of severe replacement fibrosis and lymphoplasmocytic infiltrates. Immunohystochemistry stains were strongly positive for T-lymphocyte infiltration (CD3) and weakly positive for B-lymphocytes (CD79). Polymerase chain reaction was positive for Bartonella spp. Based on these results, a post-mortem diagnosis of bacterial inflammatory cardiomyopathy was made.

Detection of Rickettsia aeschlimannii and Rickettsia africae in ixodid ticks from Burkina Faso and Somali Region of Ethiopia by new real-time PCR assays.

In the framework of cooperation for development projects in Burkina Faso and Ethiopia, we collected ixodid ticks from cattle, small ruminants and camels. We optimized new TaqMan Probe real-time PCR assays to detect Rickettsia aeschlimannii and Rickettsia africae OmpA gene in the collected samples. Rickettsia africae was identified in 75.0% Amblyomma variegatum (95%CI: 56.6-88.5), while R. aeschlimannii in 24.0% Hyalomma truncatum (95%CI: 9.4-45.1) and 50.0% H. rufipes (95%CI: 29.9-70.0) collected from cattle in different provinces throughout Burkina Faso. Ticks from the Libaan zone, Somali Region of Ethiopia, were also infected by R. africae (28.5% prevalence in Amblyomma gemma, 95%CI: 14.7-46.0) and R. aeschlimannii (27.0% H. truncatum, 95%CI: 5.0-62.9; 88.3% H. rufipes, 95%CI: 60.5-99.3). All tested ticks were adults. The developed diagnostic tools were highly sensitive and enabled us to rapidly classify R. aeschlimannii and R. africae, which were identified in Burkina Faso and in the Somali Region of Ethiopia for the first time. Further studies are needed to assess the zoonotic risk and prevalence of infection in local human populations, who have high contact rates with ticks and their animal hosts.

Evaluation of antimicrobial activity of Italian honey for wound healing application in veterinary medicine.

INTRODUCTION: Honey as a topical treatment for infected wounds dates back to ancient times. However, few studies have been reported concerning the medical properties of Italian honey. In this study, the microbial contamination, the antimicrobial activity and the antibiotic residues of 6 different varieties of Piedmont honeys were evaluated. The antimicrobial activity of honeys was tested by agar well diffusion method and 1 honey for each variety has been selected and tested by broth micro-dilution test to determine Minimum Inhibitory Concentrations (MICs) and evaluated by Minimum Bactericidal Concentrations (MBCs). The honeys with a high level of antibacterial activity were analyzed for the presence of tetracyclines, sulfonamides and macrolide residues. The agar well diffusion method showed the greatest antimicrobial activity for honeydew, chestnut and lime tree honeys. The MICs and MBCs identified the close similarity to the medical manuka honey of honeydew, polyfloral and chestnut honey. The levels of antibiotic residues on these honeys were below the limit of quantification. Based on our results the Italian variety of honeydew showed the best antimicrobial activity and can be considered for the treatment of infected wounds in animals.

INTRODUCTION: L'utilisation du miel pour le traitement des plaies infectées remonte à loin dans l'antiquité. Dans le présent travail, on étudie les contaminations microbiennes, l'activité antimicrobienne et les résidus d'antibiotiques dans 6 sortes de miels différentes provenant du Piémont. L'activité antimicrobienne a été mesurée au moyen d'une méthode de diffusion sur gel d'agar et un échantillon de chaque sorte de miel a été examiné quant à sa concentration minimale inhibitrice (CMI) et sa concentration minimale bactéricide (CMB) au moyen d'un test de micro-dilution. Les échantillons présentant une haute activité antibactérienne ont été analysés quant à la présence de tétracycline, de sulfamidés et de macrolides. Au test de diffusion sur agar, le miel de miellat ainsi que ceux de châtaignier et de tilleul ont démontré la plus grande activité antimicrobienne. Les CIM et CBM permettent de reconnaitre une grande similitude entre les miels de miellat, de nectar et de tilleul avec le miel de Manuka utilisé à des fins thérapeutiques. Les résidus d'antibiotiques de ces échantillons se situaient en dessous des limites de détection. Sur la base de ces constatations, les divers miels de miellat italiens présentent la plus grande activité antimicrobienne et peuvent être utilisés pour le traitement de plaies infectées chez les animaux.

Pathological complete response in a patient affected by multiple synchronous, breast and lung primary malignancies: a case report and review of the literature.

A pathological complete response in a patient affected by multiple synchronous, breast and lung primary malignancies is reported. A 63-year-old woman presented with an invasive ductal carcinoma of the breast and a lung adenocarcinoma. After multidisciplinary discussion, the patient underwent pulmonary left lower lobectomy followed by radio-chemotherapy with cisplatin and vinorelbine and started hormone therapy with letrozole. Ten months later, a left mastectomy with axillary lymph nodes dissection was performed. Histologically, a pathological complete response (pCR) was documented. With a review of the Literature, we discuss the issue of multiple primary malignancies, with its diagnostic and therapeutic implications. In cases of multiple synchronous malignancies it has been highlighted the importance of the choice of the best therapeutic approach for both the malignancies, reducing collateral individual effects.

Wild ungulates as sentinel of BTV-8 infection in piedmont areas.

Bluetongue caused by the genotype 8 virus (BTV-8) appeared for the first time in BTV free areas in northern Italy in 2008. The presence of domestic animals outbreaks, abundant wild ungulates populations, and ongoing regional BTV control plans, made this area interesting to evaluate the role of wild ruminants in BTV-8 epidemiology. We analyzed spleen samples from hunted red deer (Cervus elaphus), roe deer (Capreolus capreolus) and Alpine chamois (Rupicapra rupicapra) by quantitative RT-PCR. Samples were collected from 2008 to 2011 in two provinces of Piedmont region. BTV-8 was detected in all ungulate species, confirming their receptivity to the infection. However, the viral load in the positive specimens was low, and decreased from 2008 to 2011. These results, together with the extinction of the epidemic following a regional livestock vaccination campaign, lead to hypothesize that wild ungulates were an epiphenomenon and they had not an important role in the domestic transmission cycle of BTV-8 in this area. In spite of this, wild ruminants appear to be good sentinels of BTV circulation and their monitoring could be useful for surveillance in piedmont areas.

Clonal spread of Salmonella enterica serovar Infantis in Serbia: acquisition of mutations in the topoisomerase genes gyrA and parC leads to increased resistance to fluoroquinolones.

Quinolone-resistant Salmonella Infantis (n = 64) isolated from human stool samples, food and poultry during the years 2006-2011 were analysed for their resistance phenotypes, macrorestriction patterns and molecular mechanisms of decreased susceptibility to fluoroquinolones. Minimum inhibitory concentrations (MICs) of nalidixic acid (NAL) and ciprofloxacin (CIP) were determined by the agar dilution procedure, and the susceptibility to additional antimicrobial agents was determined by the disc diffusion method. To assess the influence of enhanced efflux activity, MICs were determined in the presence and absence of the inhibitor PAßN. The results of pulsed-field gel electrophoresis (PFGE) typing revealed that quinolone-resistant S. Infantis in Serbia had similar or indistinguishable PFGE profiles, suggesting a clonal spread. All S. Infantis showed combined resistance to NAL and tetracycline, whereas multiple drug resistance to three or more antibiotic classes was rare (2 isolates of human origin). The MICs ranged between 512 and 1024 µg/mL for NAL and 0.125-2 µg/mL for CIP. A single-point mutation in the gene gyrA leading to a Ser83→Tyr exchange was detected in all isolates, and a second exchange (Ser80→Arg) in the gene parC was only present in eight S. Infantis isolates exhibiting slightly higher MICs of CIP (2 µg/mL). The inhibitor PAßN decreased the MIC values of CIP by two dilution steps and of NAL by at minimum 3-6 dilution steps, indicating that enhanced efflux plays an important role in quinolone resistance in these isolates. The plasmid-mediated genes qnr, aac(6')-lb-cr and qepA were not detected by PCR assays.

Lyme borreliosis spirochetes and spotted fever group rickettsiae in ixodid ticks from Pianosa island, Tuscany Archipelago, Italy.

A study on tick fauna and tick-borne pathogens was undertaken in Pianosa, an island in the Tuscany Archipelago that constitutes an important stopping and nesting point for migratory birds. Ticks were removed from feral cats and a few terrestrial birds, and host-seeking ticks were collected by dragging. A total of 89 ticks were found on animals: 57 Ixodes ventalloi Gil Collado, 1936 and 32 Ixodes acuminatus Neumann, 1901. Host-seeking ticks were 354 Hyalomma spp. larvae and 18 Hyalomma spp. adults, identified as Hyalomma marginatum C.L. Koch, 1844 (n=11) and 7 Hyalomma detritum Schulze, 1919 (n=7). A sample of adult ticks was subjected to molecular analyses to look for Rickettsia spp. and Borrelia burgdorferi sensu lato (s.l.). Sequence analysis of the 5S-23S intergenic spacer region and OspA gene of B. burgdorferi s.l.-positive samples showed the presence of Borrelia spielmanii (n=3; 3.7%, 95% confidence interval [CI] 0.08-10.4) and Borrelia valaisiana (n=13; 13.6%, 95% CI 7.0-23.0) in Ixodes ticks from cats and terrestrial birds. Ixodes spp. were also infected by Rickettsia helvetica (n=19; 23.4%, 95% CI 14.7-34.2). Finally, we detected Rickettsia aeschlimannii in 3 out of 12 host-seeking Hyalomma spp. adults tested (25%, 95% CI 5.5-57.2). Our study shows the presence of several tick-borne pathogens in Pianosa. Hyalomma spp. and Ixodes ticks other than I. ricinus seem to be involved in their epidemiological cycle, and birds could contribute to the pathogen dispersal along their migration routes. This is the first finding of B. spielmanii in Italy. We hypothesize the involvement of peridomestic rodents or hedgehogs in its maintenance in Pianosa.

Ticks and tick-borne pathogens in livestock from nomadic herds in the Somali Region, Ethiopia.

Between May 2006 and January 2007, blood samples and ticks were randomly collected from 220 nomadic animals from Filtu and Dollo Odo districts, Libaan zone, in the Somali Region of Ethiopia. Overall, 81.5% cattle, 98.2% camels, 53.4% goats and 61.1% sheep were infested by ixodid ticks. Collected ticks (n = 1,036) were identified as Rhipicephalus pulchellus (40.1%), R. pravus (25.8%), Amblyomma gemma (9.4%), Hyalomma rufipes (13.3%), H. truncatum (2.8%), H. impeltatum (1.2%) and H. dromedarii (0.5%); immature stages (6.1%) belonged to the genera Rhipicephalus and Amblyomma. Tick infestation burden was evaluated by the Tick Abundance Score method on 57 animals from Dollo Odo in August 2006, and it was significantly higher in cattle and camels than in small ruminants (p < 0.001). Reverse Line Blot Hybridisation was applied to detect Theileria, Babesia, Ehrlichia and Anaplasma spp. Five out of 50 blood samples from Filtu, four from cattle and, surprisingly, one from a camel, were positive for Theileria mutans and two from cattle for T. velifera. Adult ticks (n = 104) from both districts were tested and A. gemma from cattle were positive to T. velifera (1) and Ehrlichia ruminantium (5 samples). Positive E. ruminantium samples were also tested by PCR targeting pCS20 and 16S rRNA genes and submitted to DNA sequencing. The phylogenetic reconstruction of pCS20 fragment showed the presence of the Somali region sequences in the East-South African group. Our results are the first available on ticks and selected tick-borne diseases from the Somali region of Ethiopia and could be used as preliminary information for planning sustainable control strategies for tick and tick-borne pathogens in the study area and in neighbouring areas with similar socio-ecological features.

LTR promoter activity of SRLV genotype E, strain Roccaverano.

The highly divergent, small ruminant lentivirus (SRLV) genotype E Roccaverano strain has a full genome consisting of 8,418 nucleotides, which lack the entire dUTPase subunit of the pol gene, the vpr-like accessory gene, and the 71-bp repeat of the U3 region within the long terminal repeat (LTR). These deletions affect in reverse transcriptase fidelity in non-dividing cells (dUTPase and vpr-like) and in the regulation of viral replication. Surprisingly, this SRLV strain was able to replicate efficiently in non-dividing cells (i.e., blood-derived macrophages), while replication in fibroblastic-like cells was somewhat restricted. To evaluate whether this observation was due to the presence/absence of specific transcription factors within these fibroblasts, U3 transcriptional activity was measured in the different cell types and revealed that both fibroblasts and macrophages were able to activate the viral promoter in the same manner. Among the transcription factor-binding sites present within the U3 region, the highly conserved Ap4 tandem repeat was shown to be sufficient for LTR promoter activity.

Serological characterization of the new genotype E of small ruminant lentivirus in Roccaverano goat flocks.

Maedi visna virus (MVV) and caprine arthritis encephalitis virus (CAEV) are a heterogeneous group of infectious agents affecting sheep and goats. Due to their natural cross-species infection they are referred to as small-ruminant lentiviruses (SRLV). Recently a new genetic cluster, highly divergent from MVV and CAEV was identified in the north-west part of Italy. A panel of genotype E specific antigens was developed and evaluated in flocks infected with B1 and E strains. The results clearly indicate that a strain specific antigen is required to correctly identify animals infected with different genotypes.

Development and application of an enzyme-linked immunosorbent assay for detection of bovine viral diarrhea antibody based on Erns glycoprotein expressed in a baculovirus system.

The BVDV envelope glycoprotein E(rns)/gp48 and the C terminal 79 amino acids of the capsid protein coding region were expressed in a baculovirus system and antigenically characterized. Western blot assay was used to detect recombinant E(rns) (r-E(rns)) in infected insect cells using specific monoclonal antibodies. The r-E(rns) was then used in an indirect ELISA to detect BVDV specific antibodies in a panel of 540 well-characterized sera. Results of the r-E(rns) ELISA were compared to those obtained with a commercially available competitive ELISA targeting anti-NS2/3 antibodies. A good correlation was observed between the 2 ELISA (kappa = 0.916, 95% C.I.: 0.876, 0.956). Using the commercial NS2/3 ELISA as the reference test, the relative sensitivity of r-E(rns) ELISA was 97.5% (95% C.I.: 94.3%, 99.1%) and the relative specificity was 93.9% (95% C.I.: 89.4%, 96.9%), while relative specificity was 100% (95% C.I.: 97%, 100%) using true negative sera (derived from a negative herd). All but 1 antigen positive animals (n = 36) tested negative in the r-E(rns) ELISA; among them all 22 confirmed PI animals were negative by r-E(rns) ELISA. The ability of r-E(rns) ELISA to identify cattle immunized with inactivated vaccine was also demonstrated in a small group of cattle, compared to an NS2/3 antibody ELISA. Results suggest that r-E(rns) ELISA represents an alternative test for antibody generated by natural infection or BVDV vaccination.

Genetic and antigenic characterization of the matrix protein of two genetically distinct ovine lentiviruses.

Small Ruminant Lentiviruses (SRLV) are a group of non-oncogenic retroviruses including Maedi-Visna virus (MVV) and Caprine Arthritis-Encephalitis virus (CAEV), which cause a chronic, multisystemic disease in sheep and goats, respectively. Phylogenetic analyses of SRLV are based in most cases on partial pol sequences. Several reports indicate that the species specificity of these viruses is not as strict as previously thought; MVV-like viruses have been found in goat populations and vice versa. Recently, the sequencing of some Italian ovine isolates has shown the presence of a new cluster more similar to classical caprine isolates (CAEV-like). Few data are available on the variability of structural proteins involved in the antibody response of infected animals. In this study, the gag gene of two genetically distinct ovine isolates, namely the MVV-like It-561 and the CAEV-like It-Pi1, was sequenced and the epitopes of matrix protein (MA) were mapped. Recombinant MAs and their subunits from both ovine aforementioned strains were tested against a panel of sheep and goat sera. Reactive epitopes were found in all three subunits of MA, although the central subunit displayed a more consistent reactivity. Epitope mapping of this subunit demonstrated that the amino acid sequence of at least one immunodominant epitope was quite different in the two strains. This antigenic variability may affect the sensitivity of a single strain-based immunoassay and suggests that both SRLV genotypes should be used in the development of future diagnostic tests, to avoid viral strain selection during the eradication programmes.

Acarological risk of exposure to agents of tick-borne zoonoses in the first recognized Italian focus of Lyme borreliosis.

Acarological risk was calculated as the probability of encountering at least one host-seeking Ixodes ricinus tick infected by the pathogen Borrelia burgdorferi sensu lato, in 100 m transects in the province of Genoa, Italy. The seasonal pattern of I. ricinus was studied using generalized estimating equations (GEE) with negative binomial error, to consider overdispersion of tick counts and repeated sampling of the same dragging sites from April 1998 to March 1999. Prevalence of infection by B. burgdorferi s.l. was evaluated by PCR and hybridization with genospecies-specific probes. Acarological risk (R) peaked in April (R = 0.2, 95% CI 0.13-0.26) and November (R = 0.29, 95% CI 0.10-0.46). Borrelia garinii and B. valaisiana were the most common genospecies at our study site suggesting a major role of birds as reservoirs. DNA from Anaplasma phagocytophilum, the agent of granulocytic ehrlichiosis in humans and animals, was amplified from an adult I. ricinus.